The human lymphocyte micronucleus assay: a comparison between whole-blood and separated-lymphocyte cultures

The induction of micronuclei (MN) by vincristine, mitomycin C and cyclophosphamide was compared in purified lymphocytes and in whole-blood cultures. With both assays, cytokinesis was blocked by cytochalasin B and MN were only scored in binucleate cells. The data suggest that whole-blood cultures may be considered a better experimental condition for the detection of MN induced by chemicals in vitro.     

NADP+-isocitrate dehydrogenase in germinating cucumber cotyledons: Purification and characterization of a cytosolic isoenzyme

The activity of NADP⁺-dependent isocitrate dehydrogenase (ICDH, EC 1.1.1.42) was investigated during the post-germinative growth of cucumber ( Cucumis sativus L. cv. Marketmore) seedlings. Isoelectric focusing showed the presence of several isoenzymes, two of which represented 70–80% of the total NADP⁺-ICDH activity in cotyledons of seedlings grown in the dark. They had pI values between 4.8 and 5.8. The isoenzyme with higher pI was purified to homogeneity by hydrophobic interaction, affinity, hydroxylapatite and anion exchange chromatography. The purified isoenzyme is a dimeric protein, consisting of two apparently identical 43-kDa subunits. It is specific for NADP⁺, inhibited by ATP and by 2-oxoglutarate, whereas it is not inhibited by citrate, succinate, and glyoxylate. The data indicate that NADP⁺-ICDH from cucumber is structurally similar to ICDHs from other plants, but it shows some peculiar biochemical characteristics.     

Culture storage age and fungal re‐isolation from host‐tissue influence Colletotrichum spp. virulence to pepper fruits

Using resistant cultivars is the most sustainable and practical approach against plant diseases. Plant germplasm and breeding lines are selected and assayed against, usually, the most aggressive or virulent strains of a pathogen (e.g., fungus) that causes the disease. However, prolong storage of the pathogen in culture media could affect virulence that, consequently, also influence the outcome of the resistance assay. This study demonstrates that long‐term storage (at least a year) of Colletotrichum truncatum and C. scovillei, causal agents of pepper anthracnose, in potato dextrose agar (PDA) medium decreased the aggressiveness and virulence of the fungus in host‐pepper fruits. However, reintroduction of the pathogen to the host and isolation of the pathogen as the new inoculum, prior to inoculation assays, increased the virulence of the fungi. These findings suggest that re‐inoculation and re‐isolation of Colletotichum truncatum and C. scovillei that have been stored for at least 1 year in PDA medium are necessary when using fungal cultures in pathogenicity and plant resistance assays to achieve desirable, comparable and reliable results.     

The human immunodeficiency virus (HIV) protease inhibitor indinavir directly affects the opportunistic fungal pathogen Cryptococcus neoformans

Highly active antiretroviral therapy (HAART), that includes human immunodeficiency virus (HIV) protease inhibitors (PIs), has been remarkably efficacious including against some opportunistic infections. In this report we investigated the effect(s) of the PI indinavir on protease activity by Cryptococcus neoformans, an opportunistic fungal pathogen responsible for recurrent meningoencephalitis in AIDS patients. Indinavir was also tested for potential effects on other parameters, such as fungal viability, growth ability and susceptibility to immune effector cells. It was found that indinavir impaired cryptococcal protease activity in a time- and dose-dependent fashion. The phenomenon was similarly detectable in ATCC/laboratory strains and clinical isolates. C. neoformans growth rate was also significantly reduced upon exposure to indinavir, while fungal viability was not affected and mitochondrial toxicity not detected. Furthermore, as assessed by an in vitro infection model, indinavir significantly and consistently augmented C. neoformans susceptibility to microglial cell-mediated phagocytosis and killing. Overall, by providing the first evidence that indinavir directly affects C. neoformans, these data add new in vitro insights on the wide-spectrum efficacy of PIs, further arguing for the clinical relevance of HAART against opportunistic infections in AIDS.     

Discordant effects of a chronic physiological increase in plasma FFA on insulin signaling in healthy subjects with or without a family history of type 2 diabetes

Muscle insulin resistance develops when plasma free fatty acids (FFAs) are acutely increased to supraphysiological levels (approximately 1,500-4,000 micromol/l). However, plasma FFA levels >1,000 micromol/l are rarely observed in humans under usual living conditions, and it is unknown whether insulin action may be impaired during a sustained but physiological FFA increase to levels seen in obesity and type 2 diabetes mellitus (T2DM) (approximately 600-800 micromol/l). It is also unclear whether normal glucose-tolerant subjects with a strong family history of T2DM (FH+) would respond to a low-dose lipid infusion as individuals without any family history of T2DM (CON). To examine these questions, we studied 7 FH+ and 10 CON subjects in whom we infused saline (SAL) or low-dose Liposyn (LIP) for 4 days. On day 4, a euglycemic insulin clamp with [3-3H]glucose and indirect calorimetry was performed to assess glucose turnover, combined with vastus lateralis muscle biopsies to examine insulin signaling. LIP increased plasma FFA approximately 1.5-fold, to levels seen in T2DM. Compared with CON, FH+ were markedly insulin resistant and had severely impaired insulin signaling in response to insulin stimulation. LIP in CON reduced insulin-stimulated glucose disposal (Rd) by 25%, insulin-stimulated insulin receptor tyrosine phosphorylation by 17%, phosphatidylinositol 3-kinase activity associated with insulin receptor substrate-1 by 20%, and insulin-stimulated glycogen synthase fractional velocity over baseline (44 vs. 15%; all P < 0.05). In contrast to CON, a physiological elevation in plasma FFA in FH+ led to no further deterioration in Rd or to any additional impairment of insulin signaling. In conclusion, a 4-day physiological increase in plasma FFA to levels seen in obesity and T2DM impairs insulin action/insulin signaling in CON but does not worsen insulin resistance in FH+. Whether this lack of additional deterioration in insulin signaling in FH+ is due to already well-established lipotoxicity, or to other molecular mechanisms related to insulin resistance that are nearly maximally expressed early in life, remains to be determined.     

Comparative analysis of the human dystrophin and utrophin gene structures

We present analysis of intronic sequences in the human DMD and UTRN genes. In both genes accumulation of repeated elements could account for intron expansion. Out-of-frame rod-domain exons have stronger splice sites and are separated by significantly longer introns as compared to in-frame exons. These features are unique for the two homologs and not shared by other spectrin superfamily genes.     

Differential microbial clearance and immunoresponse of Balb/c (Nramp1 susceptible) and DBA2 (Nramp1 resistant) mice intracerebrally infected with Mycobacterium bovis BCG (BCG)

In mice, the gene encoding Nramp1 (natural resistance-associated protein 1) exists in two allelic forms, differing for a point mutation. According to Nramp1 genotype, extensive literature documents a clear-cut distinction of inbred strains in two non-overlapping groups that phenotypically express resistance (Nramp1r) and susceptibility (Nramp1s) to systemic infections. Here, we provide evidence that Nramp1r (DBA/2) and Nramp1s (Balb/c) mice differently handle intracerebral infection with Mycobacterium bovis BCG. Distinct trends of microbial clearance from the brain and also different patterns of local immune responses occur, thus arguing on the involvement of Nramp1 gene product on the accomplishment of cerebral anti-mycobacterial defenses.